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Nanostring Analysis of Lactate Correlation Geneset

  1. Raw Data
  2. Filtering low count reads
    A. Determining Which Samples to Exclude
    B. Determining Which Genes to Exclude
  3. Compare COV of HKGs to lactate cor. genes
    A. Normalized nanostring data: comparing the COV for our 5 housekeeping genes to the COV of the genes that remained after excluding false positives
    B. RNAseq CPM data: comparing the COV for our 5 housekeeping genes to the COV of the genes that remained after excluding false positives
    C. Normalized nanostring data: Intra-assay COV for LN11 triplicate data
  4. Correlation of detected gene expression levels with 3-hour lactate levels
    A. Positively correlating genes
    B. Negatively correlating genes
  5. Correlation figures for probes that meet all of the threshold criteria
    A. Heatmap showing relative expression of the gene set detected by nanostring
    B. Plot showing showing lactate levels for each sample (red) with relative expression (grey) and mean relative expression (black) of the gene set detected by nanostring. error bars are the variance of the mean relative expression data.
    C. Mean expression between Adequate and Low performing livers for the 7 lactate correlation genes.

Summary

Of the 10 samples used, one was not usable (LV13) due to an error in sample prep. Another sample (LN11) was analyzed in triplicate to demonstrate the low level of variation between technical replicates. Data was normalized using internal positive controls and calculated in the nSolver software provided by nanostring. Housekeeping genes from the RNAseq data maintained a lower coefficient of variation then the 23 lactate correlation genes assessed. Of these 23 lactate correlation genes, only 12 yielded sufficient data above the false discovery threshold calculated using internal negative controls. Of these 12 genes, only 7 maintained a correlation of .8 with lactate levels as was observed in the RNAseq experiment.

Biomarker RNAseq fold change

Raw Data

FN12 FN11 LV12 FV13 LV13 LV11 LN13 FV12 LN11.1 LN11.2 LN11.3 FN13 LN11
ACTB 4990.04 7271.63 5190.07 3669.79 22 2786.80 3948.41 5873.95 5416.64 5196.70 5713.72 4314.44 5.442353e+03
ALB 243998.83 160622.86 186705.03 284541.81 22 93561.25 180669.25 220259.48 149218.75 141926.48 156008.36 184519.36 1.490512e+05
ATG12 166.01 160.75 134.01 104.13 22 119.25 113.05 158.46 199.39 180.39 218.10 107.61 1.992933e+02
ATG16L1 38.42 38.94 27.00 46.71 22 25.00 25.00 37.00 45.23 29.74 41.26 56.00 3.874333e+01
ATG5 214.04 151.76 203.37 153.76 22 166.62 154.92 180.97 180.92 179.40 194.52 178.99 1.849467e+02
ATP5ME 2184.26 2035.82 2116.00 1673.84 22 1411.58 1916.63 2190.57 2334.47 2125.07 2322.43 2215.97 2.260657e+03
ATP5MF 1064.69 1023.40 1050.95 786.31 22 569.20 889.75 1178.57 1008.93 965.40 1016.80 1061.86 9.970433e+02
BCL2 115.25 74.88 63.48 102.18 22 32.98 51.29 66.63 59.08 49.56 62.87 68.08 5.717000e+01
BCL2L11 16.00 78.88 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
CCL2 27.44 108.83 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
CCL4 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
CCL5 50.76 20.00 27.00 32.00 22 25.00 50.24 37.00 57.23 50.55 61.89 47.22 5.655667e+01
COX17 499.42 469.27 460.82 257.89 22 312.93 363.23 543.82 438.46 336.01 429.32 487.56 4.012633e+02
COX6B1 1355.56 1297.97 1168.50 864.17 22 811.94 924.30 1268.61 1390.16 1244.91 1377.34 1095.90 1.337470e+03
COX7B 1621.73 1975.91 1802.12 1067.56 22 1085.96 1430.93 1589.13 1487.08 1411.42 1550.25 1552.71 1.482917e+03
CPD 225.01 356.44 296.24 239.40 22 257.96 183.18 533.91 220.62 191.30 209.25 104.32 2.070567e+02
CTH 323.80 177.72 276.26 570.27 22 307.01 356.95 256.60 355.39 305.28 337.95 353.59 3.328733e+02
CXCL10 26.07 20.00 27.00 32.00 22 25.00 31.40 37.00 35.00 23.00 29.00 29.00 2.900000e+01
CYCS 21.95 31.95 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
CYP2B6 1551.76 60.90 742.95 373.69 22 138.71 13589.16 144.06 4499.09 4312.58 4553.49 18295.46 4.455053e+03
DDIT3 159.15 136.79 150.47 119.70 22 67.66 96.30 99.94 118.15 122.90 121.82 121.89 1.209567e+02
EPHX1 2004.52 755.82 1478.85 1300.14 22 796.71 5158.48 470.89 3637.86 3486.93 3854.01 6698.41 3.659600e+03
ERO1A 187.97 206.68 169.28 86.61 22 131.94 116.19 162.97 147.69 149.67 126.73 147.15 1.413633e+02
G6PC1 1151.13 914.57 1260.19 1351.72 22 1568.05 748.44 1797.12 542.77 555.05 570.78 750.00 5.562000e+02
GAPDH 5857.16 7002.05 3898.14 4071.71 22 3197.00 4328.39 7301.92 6248.33 5709.13 6354.26 4523.08 6.103907e+03
GPX2 1286.96 1028.39 1048.59 252.05 22 387.36 1831.85 460.08 2215.39 2092.36 2184.89 1580.17 2.164213e+03
GSKIP 200.32 224.65 181.04 185.87 22 146.32 129.80 192.68 130.15 136.78 149.33 96.63 1.387533e+02
GSTA1 1702.68 578.10 1015.68 4037.65 22 416.12 9807.18 142.26 13434.51 12610.64 13825.52 7987.58 1.329022e+04
GSTA2 841.05 32.95 297.42 632.56 22 25.00 6285.85 37.00 2702.78 2517.57 2735.04 3845.55 2.651797e+03
HIF1A 478.84 1180.15 285.66 188.79 22 198.76 244.94 378.15 382.16 328.08 353.67 220.72 3.546367e+02
HRCT1 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
HSP90B1 1909.85 2529.05 1749.22 241.34 22 1424.27 877.19 1358.64 884.31 854.39 997.15 1306.74 9.119500e+02
HSPA5 408.86 316.51 293.89 47.68 22 187.76 126.66 161.16 123.69 106.06 125.75 284.41 1.185000e+02
IFNG 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
IGHG3 87.81 20.00 27.00 32.00 22 25.00 86.88 37.00 155.08 143.72 151.29 74.67 1.500300e+02
IL1B 64.48 45.93 42.32 41.85 22 25.00 52.34 37.00 35.00 30.73 33.40 29.00 3.304333e+01
IL6 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
JUN 120.74 159.75 116.38 111.91 22 46.52 309.84 101.74 234.46 236.89 267.22 282.21 2.461900e+02
LDHA 4132.53 4620.78 3273.92 1574.58 22 2889.14 1486.41 4193.88 1852.62 1696.88 1869.53 1997.45 1.806343e+03
MAP1LC3A 105.65 52.92 55.25 94.40 22 56.67 73.27 69.33 69.23 63.43 96.28 94.44 7.631333e+01
MCL1 255.20 284.56 280.96 239.40 22 178.46 264.83 232.29 397.85 354.84 395.91 259.15 3.828667e+02
MZB1 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
NDUFA13 524.11 562.12 565.44 392.18 22 353.53 479.42 594.24 611.08 566.95 641.52 564.42 6.065167e+02
NDUFS6 1008.44 867.64 911.05 572.22 22 569.20 715.99 986.79 846.46 798.88 883.19 767.57 8.428433e+02
NDUFS8 552.92 540.16 463.17 341.58 22 325.62 347.53 544.72 400.62 373.67 430.30 434.85 4.015300e+02
NKG7 24.70 23.96 27.00 32.00 22 25.00 52.34 37.00 42.46 41.63 39.30 29.00 4.113000e+01
NLRP3 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
NRF1 26.07 23.96 27.00 32.00 22 25.00 52.34 37.00 38.77 28.74 52.07 66.98 3.986000e+01
OAZ1 1446.11 1067.33 1175.55 704.57 22 993.78 860.44 1385.65 1355.08 1186.43 1339.03 1124.46 1.293513e+03
PCK1 4771.89 2575.97 3594.84 4313.05 22 7782.75 2273.58 6369.14 2136.93 2016.04 2197.66 4848.11 2.116877e+03
PCSK6 1002.95 1087.30 1458.86 781.45 22 1005.62 916.97 1505.40 858.46 845.47 890.07 915.82 8.646667e+02
PMAIP1 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
PPARGC1A 104.27 87.86 195.14 165.44 22 299.40 102.58 152.16 84.92 84.25 99.22 133.97 8.946333e+01
PPBP 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
PRF1 16.00 20.00 27.00 32.00 22 25.00 26.17 37.00 35.00 23.00 29.00 29.00 2.900000e+01
PZP 2438.08 3867.95 8327.63 1415.95 22 5873.00 4034.25 6594.23 6687.72 6336.54 7001.67 6948.78 6.675310e+03
RPL13A 1406.32 1940.97 1511.76 1305.98 22 933.73 1480.13 1499.10 2161.85 2045.77 2129.87 1492.32 2.112497e+03
SERF2 28.81 20.00 27.00 32.00 22 25.00 29.31 37.00 41.54 31.72 38.31 35.14 3.719000e+01
SOCS3 50.76 72.89 48.20 32.00 22 54.97 40.82 71.13 37.85 38.66 35.37 29.00 3.729333e+01
SQSTM1 1881.04 2145.65 2741.39 1218.40 22 1518.15 2393.96 1411.76 5312.33 4886.46 5420.96 3088.96 5.206583e+03
TFAM 227.76 152.76 181.04 249.13 22 117.56 174.81 216.99 180.92 145.70 181.75 183.38 1.694567e+02
TKT 223.64 223.65 102.27 111.91 22 75.27 277.39 129.65 390.46 377.64 364.48 453.52 3.775267e+02
TMEM63A 193.45 184.71 114.03 98.29 22 128.56 38.73 243.10 62.77 65.42 66.80 105.42 6.499667e+01
TMEM92 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 43.38 41.63 29.00 29.00 3.800333e+01
TNF 16.00 20.00 27.00 32.00 22 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
TRBC1 34.30 20.97 27.00 32.00 22 25.00 30.36 37.00 35.00 28.74 31.44 29.00 3.172667e+01
WARS1 171.50 216.66 116.38 95.37 22 74.43 128.75 133.25 151.39 112.99 145.40 169.11 1.365933e+02
XBP1 2819.50 2797.63 3331.52 1832.46 22 2391.83 2180.42 4812.42 4172.32 4008.29 4134.98 2420.21 4.105197e+03
ZNF746 16.00 22.96 27.00 32.00 22 25.00 43.96 37.00 37.85 23.00 31.44 45.02 3.076333e+01
NEG_A 7.00 17.00 12.00 11.00 22 10.00 11.00 13.00 10.00 11.00 12.00 10.00 1.100000e+01
NEG_B 9.00 20.00 12.00 30.00 7 14.00 20.00 23.00 24.00 21.00 20.00 17.00 2.166667e+01
NEG_C 1.00 2.00 2.00 2.00 2 7.00 4.00 7.00 4.00 2.00 7.00 5.00 4.333333e+00
NEG_D 1.00 7.00 4.00 6.00 6 3.00 7.00 4.00 4.00 7.00 4.00 2.00 5.000000e+00
NEG_E 11.00 20.00 10.00 10.00 10 14.00 12.00 14.00 22.00 15.00 25.00 21.00 2.066667e+01
NEG_F 16.00 20.00 27.00 30.00 21 25.00 25.00 37.00 35.00 23.00 29.00 29.00 2.900000e+01
NEG_G 1.00 2.00 1.00 2.00 1 2.00 2.00 1.00 1.00 3.00 4.00 1.00 2.666667e+00
NEG_H 6.00 5.00 7.00 32.00 1 1.00 3.00 15.00 4.00 3.00 5.00 6.00 4.000000e+00
POS_A 12645.00 17909.00 15985.00 18718.00 23104 22473.00 18006.00 20221.00 20055.00 19011.00 20336.00 16846.00 1.980067e+04
POS_B 4673.00 6644.00 6209.00 6811.00 8120 8083.00 6495.00 7506.00 7123.00 6886.00 7208.00 6200.00 7.072333e+03
POS_C 1229.00 1807.00 1596.00 1888.00 2163 2294.00 1762.00 1950.00 2004.00 1944.00 1960.00 1662.00 1.969333e+03
POS_D 277.00 404.00 353.00 435.00 473 471.00 391.00 454.00 456.00 423.00 469.00 379.00 4.493333e+02
POS_E 93.00 122.00 83.00 111.00 93 93.00 104.00 140.00 117.00 108.00 105.00 94.00 1.100000e+02
POS_F 53.00 63.00 54.00 67.00 74 99.00 60.00 66.00 70.00 60.00 52.00 61.00 6.066667e+01


* Positive Controls: Each mRNA Expression CodeSet contains probes designed against fourteen ERCC transcript sequences. Six of these sequences are used as positive hybridization controls and eight are designed as negative controls. For each positive control, in vitro transcribed RNA targets are pre-mixed with the Reporter CodeSet during manufacturing. Six different transcripts are provided, each at one of the following concentrations in the 30 μL hybridization reaction: 128 fM, 32 fM, 8 fM, 2 fM, 0.5 fM, and 0.125 fM. This range of concentrations corresponds to the expression levels of most mRNAs of interest present in 100 ng of total RNA.
IMPORTANT Positive controls only account for variation hybridizaiton and recovery efficiency between lanes, not differences in total mRNA loaded!

* Negative Controls: NanoString includes several probes in each CodeSet for which no target is expected to be present. These negative controls can be used to estimate background values in your experiment, but may not capture minor probe-specific differences in background for every probe in a CodeSet.

Filtering low count reads

Excluding any probes that did not exceed the maximum count observed in the negative controls for that sample. This is the most stringent false discovery identification method suggested by nanostring

A) Determining Which Samples to Exclude

  • Excluding LV13 since none of the reads were greater than the negative control threshold

B. Determining Which Genes to Exclude

  • Excluding all genes with more than 2 false positives

Compare COV of HKGs to lactate cor. genes

Housekeeping genes should have generally lower coefficients of variation compared to the lactate correlation genes

A. Normalized nanostring data: comparing the COV for our 5 housekeeping genes to the COV of the genes that remained after excluding false positives

B. RNAseq CPM data: comparing the COV for our 5 housekeeping genes to the COV of the genes that remained after excluding false positives

C. Normalized nanostring data: Intra-assay COV for LN11 triplicate data

Correlation of detected gene expression levels with 3-hour lactate levels

Excluding any gene that does not have a correlation of at least .8 between the nanostring data and 3-hour lactate levels

A. Positively correlating genes


Note: GSTA1 is only able to meet the cutoff when rounded. (r= 0.7977059)

B. Negatively correlating genes


There are 7 genes that meet the correlation cutoff of .8 that was previously used with the RNAseq data results. All of these genes positively correlated with lactate, with no negatively correlating genes having sufficient levels of correlation. Alternatively, setting the cutoff more leniently to .65 would still only yield a total of 10 genes which would include a single negatively correlating gene

Correlation figures for probes that meet all of the threshold criteria

A. Heatmap showing relative expression of the gene set detected by nanostring

B. Plot showing showing lactate levels for each sample (red) with relative expression (grey) and mean relative expression (black) of the gene set detected by nanostring. error bars are the variance of the mean relative expression data.

C. Mean expression between Adequate and Low performing livers for the 7 lactate correlation genes.